Aims: In this study, we have investigated the biochemical behaviour of Aspergillus sp. (five strains) and Penicillium expansum (one strain) fungi cultivated on waste cooking olive oil. The production of lipid-rich biomass was the main target of the work. In parallel, the biosynthesis of other extracellular metabolites (organic acids) and enzyme (lipase) and the substrate fatty acid specificity of the strains were studied. Methods and Results: Carbon-limited cultures were performed on waste oil, added in the growth medium at 15 g l-1, and high biomass quantities were produced (up to c. 18 g l-1, conversion yield of c. 1 center dot 0 g of dry biomass formed per g of fat consumed or higher). Cellular lipids were accumulated in notable quantities in almost all cultures. Aspergillus sp. ATHUM 3482 accumulated lipid up to 64 center dot 0% (w/w) in dry fungal mass. In parallel, extracellular lipase activity was quantified, and it was revealed to be strain and fermentation time dependent, with a maximum quantity of 645 U ml-1 being obtained by Aspergillus niger NRRL 363. Storage lipid content significantly decreased at the stationary growth phase. Some differences in the fatty acid composition of both cellular and residual lipids when compared with the initial substrate fat used were observed; in various cases, cellular lipids more saturated and enriched with arachidic acid were produced. Aspergillus strains produced oxalic acid up to 5 center dot 0 g l-1. Conclusions: Aspergillus and Penicillium strains are able to convert waste cooking olive oil into high-added-value products. Significance and Impact of the Study: Increasing fatty wastes amounts are annually produced. The current study provided an alternative way of biovalourization of these materials, by using them as substrates, to produce added-value compounds.